Members of the ARF family of regulatory GTPases function as nodes in cell signaling to coordinate essential cell processes; including membrane traffic, energy metabolism, and the cytoskeleton. The focus of this application is one of those GTPases, ARL2, and its binding partners. An understanding of the molecular mechanisms of ARL2 action to carry out or regulate essential cell processes will reveal novel insights into fundamental aspects of cell biology as well as providing potential targets for intervention to alte the course of human diseases; including but not limited to cancer, heart disease, neurodegeneration, retinal degeneration, and deafness. During the previous four years of funding we have provided rigorous tests of several models for ARL2 actions in different parts of eukaryotic cells, discarding some and refining others. We have also generated many key reagents, including the first purified protein preparations of mg levels of four different proteins and a series of point mutations in ARL2 that allow dissection of its different essential roles in eukaryotic cells. Thus, we are poised to make much more rapid progress in our understanding of the molecular mechanisms of regulation of tubulin folding and polymerization, ATP generation in mitochondria, and mitochondrial fission and motility. In the next funding period we propose three new, specific aims that are logical extensions of our previous work but which promise to have a far broader impact on several fields of cell signaling and regulation. In aim 1 we will test the model that ARL2 acts with the tubulin-specific co-chaperone cofactor D (TBCD) to regulate tubulin folding, microtubule density or polymerization, and less directly cell division. In aim 2 w will test the hypothesis that ARL2 regulates ATP production in mitochondria via changes in cristae remodeling and mitochondrial fusion. And in aim 3 we examine how mitochondrial fusion and motility are regulated by the ARL2 effector and GAP, ELMOD2.